Purification and specific kinetic properties of erythrocyte uridine diphosphate glucose pyrophosphorylase.

The enzyme uridine diphosphate glucose pyrophosphorylase has been prepared at high purity (specific activity 127) from the human erythrocyte. Comparison of its specific properties with enzyme isolated from liver indicates many similarities including size. Examination of the reversible reaction catalyzed by the erythrocyte enzyme from both forward and reverse directions revealed a highly selective product inhibition by UDP-glucose. Since a dual enzyme function involving both regulation and synthesis of UDPglucose was implied, additional UDP-glucose pyrophosphorylases were examined in relation to their product inhibition characteristics for comparative purposes. The product inhibition studies were extended and applied in distinguishing the kinetic mechanism of erythrocyte UDPglucose pyrophosphorylase. From the product inhibition patterns and initial velocity studies an ordered Bi-Bi reaction mechanism was indicated in which nucleotide adds first as substrate to the enzyme and is last to be released. Michaelis constants for each of the substrates and dissociation (inhibition) constants for substrates combining with free enzyme were also determined.